Cell wall chemical characteristics of whole-crop cereal silages harvested at three maturity stages.

BACKGROUND: In cooler climates such as found in Scandinavian countries cereals are important feedstuffs for ruminants often ensiled as whole-crop cereal silages (WCCS) to preserve nutrients. Animal performance varies with the type of cereal forage and stage of cereal development being ensiled. Cell wall isolation and analysis was undertaken to determine differences among cereal silages harvested at different stages of maturity.
RESULTS: A set of 27 WCCS samples of barley, wheat and oats harvested at heading, early milk, and dough stages of maturity were analyzed for cell wall (CW) composition and compared to previous NDF analyses. Total CW concentrations of the WCCS were higher than the NDF concentration. The lignin concentration was higher (P < 0.001) in oats (111 g kg(-1) DM) than in barley (88 g kg(-1) DM) and wheat (91 g kg(-1) DM). Ferulates (ester and ether linked) ranged from 12.2 to 14.9 g kg(-1) across forage types and maturity stages. The correlation between total cell wall xylose and HC concentrations (NDF-ADF) was lower than expected in all forages (R = 0.63).
CONCLUSION: The more comprehensive analyses of cell walls provide detailed composition of the different WCCS that vary due to the maturity and type of cereal.

INTRODUCTION

High‐producing dairy cows require diets that contain sufficient nutrients to support the desired levels of milk production and body maintenance. Producing forage crops that meet nutrient requirements is a challenge especially in regions of northern latitudes with restricted growing season limiting the range of cropping alternatives. Cereals such as wheat, barley, and oats are well suited for production in cooler shorter growing seasons found in Sweden.1, 2 Production of cereals in cooler environments can be challenging since the goal is to maximize total nutritive value.3 Cell wall fractions (fiber) increase and generally have decreasing nutritive value due to decreased digestibility while continued development results in starch production that generally increases nutritive value.

With increasing maturity total biomass increases but digestibility decreases and is most often associated with increased levels of lignin.4 This relationship holds within a given species, but may not work so well across species even when comparing similar stages of development.5 In the Poaceae family (grass family) the hydroxycinnamates, p‐coumaric acid (pCA) and ferulic acid (FA) influence digestibility. For pCA they are esterified primarily to sinapyl alcohol but also to coniferyl alcohol and become part of the lignin structure as the monolignols undergo radical mediated polymerization.6 The pCA does not become incorporated but remains simply esterified to the growing lignin polymer.7 To a lesser extent pCA can also be incorporated ester linked to arabinosyl (Ara) units of arabinoxylans.8 Ferulates are incorporated into the cell wall esterified to arabinosyl residues of arabinoxylans9 and form cross‐links with other ferulates10 as well as incorporation into growing lignin polymers.11 In grasses the digestibility of cell walls is dependent not only on the amount of lignin but also the degree of cross‐linking. Cross‐linking can be between arabinoxylans as well as arabinoxylans and lignin polymers both having a negative impact upon digestibility.12, 13

The detergent system was developed as a rapid method of estimating nutritive value of forages and other feedstuffs and works well for this purpose.14, 15 However, measuring lignin with the detergent system can be a challenge. It has been clearly demonstrated that the typical detergent method, acid detergent lignin (ADL), can lead to under‐estimation of lignin in grass cell walls. Hot detergent solutions, especially acid detergent, solubilizes lignin from the cell wall matrix of grasses.16, 17 The detergent system may not reveal sufficient detail about the chemical make up of certain types of forages to provide a clear picture of how chemical composition is related to animal performance. This study was undertaken to determine the chemical composition obtained from a complete cell wall analysis of three different whole cereal crop silages (WCCS).

MATERIAL AND METHODS

Silages

A total of 27 samples of nine different WCCS from two feeding experiments with dairy heifers were analyzed.1, 2, 18 The WCCS used were oats (Avena sativa L.), six‐rowed barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) (all Swedish varieties) harvested at the heading, early milk and early dough stages of maturity. The WCCSs were ensiled in plastic wrapped big bales. Three bales were selected from each stage of development for each cereal. Multiple cored subsamples were taken from each bale and combined to create the replicate sample for analyses.1, 18, 19

Chemical analyses

Cell wall extraction

Approximately 1.5–1.7 g of sample was accurately weighed into 40‐mL Oakridge tubes on a dry matter (DM) basis (55 °C). All samples were extracted as outlined in the cell wall extraction flow chart (Scheme 1). Final cell wall concentration was determined after drying at 55 °C for at least 24 h and was expressed on an ash‐free basis (CWom). Determinations of cell wall components were based upon the isolated cell wall residue. Cell wall residues were oven‐dried at 55 °C for 24 h before weighing subsamples for the different cell wall analytical procedures.

Scheme 1

Comparison of the extraction methods to produce (A) total cell wall and (B) NDF components from cereal whole crop silages.

Carbohydrate analysis

Neutral sugar components were determined as alditol acetates of the released sugars following acid hydrolysis. Cell wall residues were hydrolyzed using the Saeman method20 as modified by Hatfield et al. 17 Accurately weighed (∼100 mg) samples were subjected to a two‐stage hydrolysis: stage 1, 12 mol L−1 H2SO4, (2 h, 22–24 °C); stage 2, acid was diluted to 1.5 mol L−1 with dH2O, capped tightly and placed in a 100 °C forced air oven for 3 h. After hydrolysis, samples were cooled in an ice waterbath, centrifuged (900× g) for 10 min and 200 μL removed from each for total uronosyls determination and inositol was added as internal standard. Sub‐samples were neutralized with barium carbonate, clarified by centrifuging (3200 × g, 15 min) and filtered through a glass fiber filter (0.2 µm, Acrodisc). Sub‐samples were dried and sugars converted to alditol acetate derivatives using the procedure of Blakeney et al. 21 and analyzed by FID‐GLC (Supelco, Bellefonte, PA USA; SPB‐225 column 30 m × 0.25 mm with 0.25 µm film thickness) using a temperature program of 215 °C initial for 2 min, 4 C min−1 to 230 °C and hold for 11.25 min.

Total uronosyls in the cell wall hydrolyzates were determined by colorimetric assay following the method of Blumenkrantz and Asboe‐Hansen.22 The 200‐μL aliquots removed from the cell wall hydrolyzate were individually diluted to 2 mL using dH2O and this diluted sample was used in the assay.

Lignin determination

Acetyl bromide lignin (ABSL) was measured following the procedure of Morrison23 as modified by Hatfield et al. 24 Dry cell wall samples of 20–25 mg were weighed into Pyrex tube (16 mm × 200 mm) fitted with a Teflon lined cap, suspended in 2.5 mL of a 25% acetic bromide in glacial acetic acid and heated for 2 h in a heating block at 50 °C. The samples were mixed every 20 min during heating. The absorbance maximum between 275 nm and 280 nm was determined by evaluating spectral scans (250–350 nm) for each sample.

Cell wall phenolic determinations

Ester and ether linked ferulic acid and p‐coumaric acid, were analyzed using the sequential method.25 Phenolics were identified and quantified as trimethylsilane derivatives (40 μL TMSI; Thermo Scientfic, Rockford, IL, USA and 10 μL pyridine) using GLC‐FID on a ZB‐5 ms column (Zebron; 30 m × 0.25 mm, 0.25 µm film). The GLC conditions were injector 315 °C, detector 300 °C, and a temperature program of 150 °C for 5 min, 4 °C min−1 to 200, 10 °C min−1 to 240 °C, 30°Cmin−1 to 300 °C and hold for 10 min.

Nitrogen and ash

All cell wall samples were analyzed for total N using a combustion assay (Leco FP‐2000 N Analyzer; Leco Instruments, Inc., St. Joseph, MI, USA) and they were also analyzed for ash by combustion at 500 °C.

Detergent analyses and permanganate lignin

Detergent fiber fraction information (NDFom, ADF, ADL) was complied from previous work on the same cereal silages.1, 2, 18

Statistical analysis

The statistical analysis was done with the mixed procedure in SAS version 9.2 (SAS Institute, Cary, NC, USA). The initial model for all chemical components was:

where Y is the general mean, C is the fixed factor of cereal species, M is the fixed factor of maturity stage at harvest, C*M is the crop (C) × maturity (M) fixed interaction factor of cereal species and maturity stage at harvest, and e is the random error. If the C*M interaction factor was significant (P < 0.05), LS means for C*M was tested with the PDIFF statement in SAS, and the difference between the LS means was adjusted with Tukey's test. If the C*M interaction effect was near significance (0.10 > P > 0.05) it was kept in the model, but not evaluated and if it was not significant (NS, P > 0.10) it was removed from the model. The LS means of significant single factors C and M were evaluated with the PDIFF statement in SAS and the differences were corrected with Tukey's adjustment. In all situations differences between LS means was considered significant at P Tukey's < 0.05. Linear regressions and correlations between chemical fractions were analyzed with the regression procedure and the CORR option in SAS version 9.2 and correlations were considered significant at P < 0.05.

RESULTS

Cereal species

Table 1 provides the fermentation characteristics of the individual silages chemically characterized in this study. All silage samples were dried at 60 °C and ground in a hammer mill with 1‐mm sieve.1, 2, 18 More detailed information about the experiment with the oat and six‐rowed barley can be found in Wallsten et al.,1, 19 and about the experiment with the wheat silages in Rustas et al. 18 The average detergent composition and lignin concentration for nine silages can be found in Table 2.

Table 1

Fermentation characteristics of silages evaluated in this study

Variable	Barley			Oats			Wheat
	Heading	Milk	Early dough	Heading	Milk	Early dough	Heading	Milk	Early dough
pH	4.6	4.4	4.5	4.5	4.1	4.6	4.1	4.6	5.5
NH3‐N (g kg−1 N)	71	70	67	131	72	58	72	72	54
Lactic acid	43	31	29	61	73	38	63	22	12
Acetic acid	8	11	8	20	14	8	14	13	5
Propionic acid	1.2	1.5	1.1	1.9	1.2	1.2	<1	<1	<1
Ethanol	5.3	2.9	2.6	4.5	2.8	3.4	5.5	3.8	6.9

Data complied from previous work, with permission (Rustas et al. 18 and Wallsten et al. 1).

All values are on a g kg−1 DM basis except for the NH3‐N.

Table 2

Detergent composition and lignin content measured in nine different whole‐crop cereal silages

Component	Barley			Oats			Wheat			Cereal			Maturity stage
	Head.	Milk	Dough	Head.	Milk	Dough	Head.	Milk	Dough	Barley	Oats	Wheat	Head.	Milk	Dough
NDFom (g kg−1 DM)	500	433	411	527	530	442	539	487	466	448	500	497	522	483	440
ADF (g kg−1 DM)	307	277	263	346	346	289	347	325	311	282	327	328	333	316	288
PMlignin (g kg−1 DM)	44	53	51	56	60	52	51	57	61	49	56	56	50	57	55
Hemicellulose (g kg−1 DM)	193	156	148	181	184	153	191	162	156	166	173	170	188	167	152
Cellulose (g kg−1 DM)	263	224	212	290	286	237	296	269	249	233	271	272	283	260	233
PMlignin (g kg−1 NDFom)	88	122	124	106	113	118	95	116	132	110	112	113	96	117	125
Hemicellulose (g kg−1 NDFom)	386	360	360	343	347	346	355	332	334	370	346	341	361	346	346
Cellulose (g kg−1 NDFom)	526	517	516	550	540	536	550	552	535	520	542	546	542	537	529

Data are summarized from previously published work, with permission (Rustas et al. 18 and Wallsten et al. 1).

DM = dry matter, NDFom = ash‐free neutral detergent fiber, ADF = ash‐free acid detergent fiber, PMlignin = permanganate lignin, hemicellulose = NDFom‐ADF, cellulose = ADF‐PMlignin.

The CWom concentrations were higher in oats than in barley and wheat at all maturity stages (Table 3 and Table 4). The ash concentration was highest in barley silage. Total protein retained in the cell wall fraction (CP g kg−1) was highest in barley, intermediate in oats and lowest in wheat (Table 4) corresponding to wheat having the lowest CP. Total lignin as ABlignin concentration was higher in oats than in barley or wheat. Permanganate lignin (PMlignin) performed on ADF residues resulted in lower total lignin values compared to ABlignin (Table 2 and Table 3). As a consequence PMlignin represented a fraction of the total lignin as measured by ABlignin in all the silages with oats having the lowest fraction of lignin (Tables 3 and 4).

Table 3

Ash‐free cell wall (CWom) concentration (g kg−1 dry matter) and composition (g kg−1 CWom) of three cereals harvested and stored as whole‐crop silage at three maturity stages (n = 9)

Sample	Cereal (C)			Maturity (MS)			SEM	Significance
	Barley	Oat	Wheat	Heading	Milk	Dough		C	MS
CWom	534a	588b	534a	581b	531a	543a	4	***	***
Ash	104b	80ab	68a	94	86	72	8.5	*	NS
Ash (g kg−1 of total ash)	440	400	420	430	420	420	26	NS	NS
CP in CW	73c	62b	43a	56a	56a	66b	1.2	***	***
CP (g kg−1 of total CP)	340b	340b	210a	260a	260a	380b	7.0	***	***
Ablignin	166a	189b	171a	168a	183b	175b	3.0	***	*
PMlignin (g kg−1 ABlignin)	560ab	510a	620b	520a	590b	580ab	2.0	**	*
pCA	4.8a	10.1b	5.1b	7.6c	6.8b	5.6a	0.1	***	***
FA ester	6.9b	7.0b	6.5a	7.6b	6.6a	6.3a	0.1	**	***
FA ether	7.3	6.7	6.4	6.6	7.2	6.6	0.3	NS	NS
UA	24.4ab	23.0a	28.5b	25.6	25	25.3	1.5	NS	NS
HC sugars	389b	368a	358a	393c	374b	349a	4.6	***	***
Glucose	453	460	473	455a	460a	473b	7.4	NS	**

CP, crude protein; ABlignin, acetyl bromide lignin; pCA, p‐coumeric acid; FA, ferulic acid; UA, uronic acid; HC sugars is the sum of xylose, arabinose, fucose, galactose, mannose and rhamnose.

Ash and CP are the amounts found in the CW isolates and the ash (g kg−1 of total ash) and CP (g kg−1 of the total CP) are the totals found in the DM that was retained in the CW isolation.

Values on the same row within cereal or maturity stage with different superscripts are significantly different (P Tukey < 0.05).

NS, not significant;

P < 0.05,

P < 0.01,

P < 0.001.

Table 4

Evaluation of the significant cereal*maturity stage interaction for nine different whole crop cereal silages (n = 3)

Sample	Heading stage			Milk stage			Dough stage			SEM	C*MS
	Barley	Oat	Wheat	Barley	Oat	Wheat	Barley	Oat	Wheat
CWom	563a	607b	575ab	507a	587b	497a	530a	569b	530a	6.9	**
Ash	111	107	63	113	75	70	86	59	71	15.5	NS
Ash (g kg−1 of DM ash)	430	460	390	460	380	410	430	360	470		NS
CP	67b	55a	46a	74c	53b	41a	79b	78b	42a	2.0	***
CP (g kg−1 of DM CP)	290b	300b	190a	300b	270b	190a	420b	460b	260a	12.0	***
Ablignin	164	183	157	167	199	181	165	184	175	4.9	NS
PMlignin (g kg−1 ABlignin)	480	500	570	620	520	630	590	500	660		NS
pCA	6.1a	11.6b	5.0a	4.6a	10.7b	5.2a	3.8a	8.0b	5.0a	0.2	***
FA ester	8.6b	7.2a	6.9a	6.5ab	7.1b	6.2a	5.7a	6.7b	6.4ab	0.2	***
FA ether	6.3	6.5	6.9	8.3	7.0	6.4	7.3	6.6	5.8	0.5	NS
UA	26.2	22.1	28.5	23.2	23.5	28.2	23.7	23.3	28.9	2.9	NS
HC	420	383	374	384	378	360	364	344	338	7.9	NS
Glucose	450	440	475	431	463	458	476	487	486	13.3	NS

All values are in g kg−1 CW.

CP, crude protein; ABlignin, acetyl bromide lignin; pCA, p‐coumeric acid; FA, ferulic acid; UA, uronic acid; HC sugars is the sum of xylose, arabinose, fucose, galactose, mannose and rhamnose.

Ash and CP are the amounts found in the CW isolates and the ash (g kg−1of total ash) and CP (g kg−1 of the total CP) are the % of the totals found in the DM that was retained in the CW isolation.

Values on the same row within heading/milk/dough/barley/oats or wheat with different superscripts are significantly different (P Tukey < 0.05).

C*M type of cereal silage (C) by maturity (M).

Concentrations of pCA were nearly double in oats compared to wheat or barley at all maturity stages (Tables 3 and 4). The difference in total ester‐linked FA (monomer and dimer) concentration among cereals varied with maturity stage, with highest concentration in barley at heading. Concentrations of total FA were higher in oats than barley at dough stage (Table 4). Wheat had lower concentrations of ester‐linked FA at most stages of development. Ether‐linked FA varied from 5.8 to 8.3 g kg−1 CWom (monomer + dimer), but there were no significant differences among cereal species at any maturity stage (Table 4).

Cereals and grasses in general have low amounts of pectins (<2%) with the bulk of cell wall carbohydrates distributed between hemicelluloses and cellulose.26, 27 Total uronic acids composed of both galacturonosyls and glucuronosyls were higher in wheat than in oats and tended (P Tukey = 0.071) to be higher in wheat than in barley (Table 3) though these did not account for a major portion of the cell wall carbohydrates. Hemicellulosic sugars (primarily arabinoxylans) concentrations were higher in barley than in oats and wheat. Glucose concentrations in wheat tended (P Tukey = 0.066) to be higher than barley but not significantly different from oat silage cell walls (Table 3).

Effect of maturity stages

CWom concentrations were higher at the heading stage for all three‐cereal species (Tables 3 and 4). Differences between milk and dough stage CWom were not large enough to be significant. The higher cell wall residual crude protein (rCP) concentration at dough stage was only evident for barley and oats (Tables 3 and 4). For wheat there was a marginal decrease in rCP concentration with later maturity stage (Table 4). The CP at dough stage was more difficult to remove during the washes and the percentage of rCP retained was higher at dough stage for all cereal species (Tables 3 and 4).

ABlignin concentration was lower at heading compared to at milk and dough stages. Moreover, the PMlignin at milk stage represented, and at dough stage tended to represent (P Tukey = 0.069), a higher portion of the corresponding ABlignin, than PMlignin at heading. The pCA concentration decreased with maturity for barley and oats, but for wheat the concentrations were similar and the highest value was at milk stage (Tables 3 and 4). The concentration of ester‐linked FA in barley decreased with maturity stage and a similar trend might be suggested for oats, though the numerical difference was small (7.2 to 6.7 g kg−1). The HC sugars decreased with later maturity stage, but glucose actually increased between milk and dough stage (Table 3).

Correlation of CWom to the detergent system and permanganate lignin

CWom values were generally higher than the corresponding NDFom values (Fig. 1A). Wheat at milk stage was the only silage where NDFom and CWom values were similar, and excluding it increased the positive correlation between the fiber fractions from 0.63 to 0.76. The correlation between NDFom and CWom increased when splitting the dataset on cereal species, whereas splitting it on maturity stages increased the correlation only for the samples harvested at milk stage (Table 5). Generally, as CW increased, NDF increased across all types of silages (Fig. 1A). The correlation between HC NS sugars and HCNDF was significant, but the variation in HC NS values among the replicates was large for most of the silages (Fig. 1B; Table 5). The correlation improved only for wheat when splitting the dataset on cereal species. Glucose and cellulose was not significantly correlated for the whole dataset or for the cereal species (Table 5). However, when splitting the data on maturity stages the correlation was significant for heading and milk stage. There was a large variation in glucose concentration among replicates that was not evident for corresponding cellulose concentrations when considering the mean of replicates (Fig. 1C). The PMlignin was not significantly correlated to the ABlignin, when looking at the whole dataset. Splitting it on cereal species resulted in positive correlation for oats, but negative correlation of similar magnitude for barley. For barley the ABlignin was the same irrespective of PMlignin values (Fig. 1D).

Figure 1

Correlations between chemical fractions in barley, oats, and wheat harvested at heading, milk stage, and dough stage of maturity. All comparisons are between results of the detergent analysis system and the cell wall isolation system on an ash free basis. (A) Total NDF versus total cell wall organic mater (CW); (B) detergent permanganate lignin (PMLignin) versus acetyl bromide lignin (ABLignin) determination in cell wall isolates; (C) NDF‐ADF hemicellulose versus cell wall hemicellulose based on neutral sugar analysis(HC NS); (D) ADF‐lignin cellulose versus total glucose from neural sugar analysis of cell walls. All points are the mean of three separate samples. Error bars indicate the SD of the mean and all values are on a ash free dry matter basis. OH, oats at heading; OM, oats at milk stage; OD, oats at dough stage; BH, barley at heading; BM, barley at milk stage; BD, barley at dough stage; WH, wheat at heading; WM, wheat at milk stage; WD, wheat at dough stage.

Table 5

Correlation and linear regression statistics for different chemical fractions in nine different whole crop cereal silages

Sample	CWom = NDFom			ABlignin = PMlignin			HC sugars = HC			Cellullose = glucose
	Corr	RMSE	P value	Corr	RMSE	P value	Corr	RMSE	P value	Corr	RMSE	P value
All samples	0.63	29.5	<0.001	0.27	11.7	0.167	0.65	16.8	<0.001	0.35	28.4	0.077
Barley	0.69	20.5	0.040	−0.76	4.3	0.164	0.68	17.9	0.44	0.2	24.8	0.61
Oats	0.81	13.5	0.008	0.75	6.1	0.02	0.65	14.6	0.56	−0.57	24.7	0.112
Wheat	0.73	24.7	0.26	0.55	1.8	0.125	0.9	8.6	0.001	0.41	20.2	0.277
Heading	0.43	21.7	0.253	0.50	10.2	0.171	−0.053	14.1	0.893	0.67	14.4	0.049
Milk	0.74	30.8	0.022	0.49	14.6	0.18	0.505	17.7	0.166	0.74	20.0	0.023
Dough	0.26	24.1	0.51	−0.09	10.4	0.823	−0.455	8	0.219	0.13	18.9	0.735

Total observations N = 27, total silages three species × three maturities, n = 9.

NDFom, ash‐free neutral detergent fiber; ADF, ash‐free acid detergent fiber; ABlignin, acetyl bromide lignin; PMlignin, permanganate lignin; HC (hemicellulose) = NDFom‐ADF; HC sugars = sum of xylose, arabinose, fucose, galactose, mannose and rhamnose; Cellulose = ADF‐PMlignin.

DISCUSSION

Based on the information complied in Table 1 all the silages appeared to ferment reasonably well providing a good source of animal feed. It is also clear that fermentation at the later maturities was not as robust as earlier stages most likely due to a decrease in readily available soluble sugars.1, 3 The CW analysis system provides more detailed chemical information about the fiber fraction of plants. Neutral detergent fiber (NDF) method was developed to rapidly estimate the nutrient value of forages and other feedstuff. It is intended to represent the more slowly digested CW material in a forage sample. Typically for grasses there is a good correlation between NDF (Table 2) and the CWom (Tables 3 and 4). Grasses tend to produce NDF values aligned with CWom compared to legumes primarily due to the low levels of pectins in grasses.27 As forages mature there is an increase in the CWom as a proportion of the total DM. However, in the case of cereals advanced maturity results in the formation a grain head that contributes to a significant increase in overall DM content. For this study WCCS CWom and consequentially NDF decreases as a portion of the DM with increased maturity due to rapid accumulation of starch during grain head development boosting the overall DM content. Differences among the types of WCCS for CWom are due to compositional and structural changes during maturation.28 For this work the non‐cellulosic sugars were combined into one group referred to as the hemicellulosic sugars. Although the detergent system creates an NDF fraction that does a reasonable job of representing the CW fractions there are significant differences between the two methods.

The CW isolation method (Scheme 1A) is designed to preserve all the CW components in the final insoluble residue while removing as much of the non‐CW components as possible. With no detergent being used in the extraction protein removal is less efficient than with the NDF method (Scheme 1B). The CP recovery in the CWom in the present study ranged from 190 to 420 g kg−1 (Table 4) of the original CP in the DM. Acosta et al. 29 reported a 113–157 g kg−1 recovery in barley silage harvested at the boot and the dough stages. Coblentz et al. reported levels of 250–320 g kg−1 (in NDF)30 for wheat and oat whole crops harvested between the heading and the dough stages but not ensiled. Higher CP was recovered in the NDF fraction at later maturity stages as was seen for CP in CWom in this study (Table 4).29 Increases in cell wall associated protein might be related to a reduction in apparent CP digestibility at later maturity stages probably due to the slower degradation of mature CW.1, 18, 30

Differences between NDF (Table 2) and total CWom (Table 4) may also reflect losses of cell wall material during the NDF procedure, due to solubility in the hot detergent16 and to particle losses during filtering.31 At early stages of development grass arabinoxylans may be highly branched32 and therefore more susceptible to solubilization in hot detergent solution. Arabinose side chains, which are typically in the furanose form, are susceptible to weak acid hydrolysis such as the low pH conditions produced during ensiling and could be sufficient to cleave some of theses residues.33 Larger differences were seen in measuring the lignin fraction. ABlignin used in the CWom method accounts for all the lignin whereas the PMlignin method is measuring only a fraction of the total. Differences in the chemical/physical make‐up of the cereal lignins could alter their solubility in hot detergent. Lignin, especially in grasses, can be soluble in both neutral16 and acid detergent16, 34 resulting in much lower lignin values.17 Acetyl bromide lignin method was originally proposed as a rapid method for woody samples and adapted by Morrison for forages.35 The method effectively solubilizes the lignin from the cell wall matrix in an acidic medium leaving protein and complex carbohydrates insoluble. These insoluble materials are removed with centrifugation to prevent light scattering that would alter the true lignin value. Isolated and purified lignins can be used as standards to allow quantification of lignin in unknown samples.36 In this study all the WCCS had higher lignin values compared to the detergent PMlignin in the earlier studies (Table 2). Oat silages tended to have the highest ABlignin whereas wheat tends to be higher for PMlignin. The observation that cereals with the highest ABlignin did not also give the highest PMlignin values suggests there are structural and possibly compositional differences among the different lignins. These differences are due to variable solubility in hot detergent solutions especially the AD treatment and could have implications on how cell wall materials can be degraded by microbes.

Lignin content does not appear to change significantly (Table 3) within individual silages during development when expressed on a CW basis. However, lignin on a total DM basis appears to decrease (Fig. 1B) due to the accumulation of starch as a significant portion of the DM. The most significant changes occur in the forage stems as they transition from vegetative to reproductive stages. Continued development of the grain head in cereals could account for increased accumulation of cell walls associated with the grain but contain low levels of lignin. This may be particularly true for oats and barley that accumulate large amounts of mixed linked β‐glucans37 that would remain as cell wall components during the CW extraction process. To clarify this relationship it would be necessary to evaluate the individual cereals as they stand in the field and separated into major tissue types (e.g. leaves, stems, sheaths, and reproductive parts) compared to what is available after ensiling. However, this was beyond the scope of this work and is a subject for future research.

Grass pCA is primarily incorporated into CW as an ester linked conjugate with monolignols.38, 39 In this study oats had the highest pCA and ABlignin, while wheat and barley lignin concentrations were less (90 and 120 g kg−1, respectively). The pCA concentrations however, were 500 and 520 g kg−1 lower. Previous studies indicated that across species there is not a good correlation between total lignin and pCA.38 It remains unclear as to the role of pCA in CW development. It has been suggested that the formation of p‐coumaroyl‐sinapyl alcohol conjugates help in the formation of syringyl type lignins.40 Recently, the gene for the pCA‐transferase was down regulated in two different plant systems, Bracchypodium41 and corn.42 In both cases the proportion of syringyl units incorporated into lignin decreased, but the total lignin content did not decrease. It may be the formation of pCA‐sinapyl alcohol conjugates aids in the incorporation of syringyl units in lignin, but it does not control overall lignin formation.

Among the hyrdoxycinnamates, FA is most likely to influence CW digestibility, by cross‐linking arabinoxylans forming FA‐dimers. FA and FA‐dimers can also be coupled with lignin forming cross‐linked networks of arabinoxylans and lignin.10 For barley and oats ether linked FA increased or remained constant while wheat appeared to decline during maturation (Table 4). Linkage of ferulates, both monomers and dimers, to lignin is not always through an ether type linkage43 but only the ether linkages can be hydrolyzed and accounted for in this analysis.44 Therefore not all cross‐links between lignin and xylans can be accounted for in the typical analysis procedure. As already discussed barley and oat cereals incorporate relatively high levels of mixed linked β‐glucans during grain development.37 The β‐glucans are a part of the grain CW but would not be retained during hot detergent extraction. Their rapid accumulation during grain development would contribute to an overall increase in the CW fraction without increasing FA content making it seem like it is decreasing during this stage of cereal development.

The hemicellulosic (HC) fraction is typically composed of all the non‐glucose sugars, mainly arabinose and xylose with contributions from galactose, mannose, rhamnose and fucose. Glucose is primarily associated with cellulose. Exceptions are oat and barley at the grain filling stage where part of the glucose is from mixed linked β‐glucans. The correlation between CWom HC and the NDF‐ADF HC (Table 5) decreased due to a large variation between replicates. Replicates were from different bales and may be expected to have some difference in amounts. However, since the HC sugars did not differ in the same way it may instead have been a result of determining the NDF and the ADF on different subsamples instead of doing a sequential NDF‐ADF analysis. It is also possible for the hot detergent reagent for NDF to solubilize parts of the hemicellulosic fraction in these cereals resulting in variable recoveries.27 Although there is variability in the individual samples a plot of the means for WCCS samples by species and maturity stage shows a good relationship between CW HC‐NS (based on a sum of the non‐glucose sugars) and HC based NDF‐ADF (Fig. 1C). When calculated on a DM basis there is a consistent decrease in hemicellulosic sugars as the cereals mature.

The cellulose concentration of replicates based on the detergent system was more tightly clustered. The use of permanganate to degrade the lignin (PMlignin) leaving a cellulose residue may actually help remove some of the variability as a result of ND analysis (Fig. 1D). Permanganate treatment can degrade some of the non‐cellulosic carbohydrates in the CW that may remain in the ADF residue. The larger variation in the glucose content (based on CW isolation) is most likely due to the formation of mixed linked β‐glucans in the developing grain in oat and barley. Depending upon the stage of grain fill the quantity of β‐glucans could be variable as well as the solubility during the CW isolation procedure.45 Wheat has low levels of β‐glucans in the grain, but has arabinoxylans containing some ferulates.46 Loss of lignin during the NDF and ADF analyses16, 47 could result in cellulose concentration being overestimated in these samples. The improved correlations (Table 5) when the dataset was split on maturity stages could be explained by the presence of β‐glucans at dough stage, which is present in high amounts in the grains of barley and oats, but in lower amounts in wheat.48 The presence of β‐glucans might explain why the variation in glucose concentration among replicates was so much larger compared to both cellulose concentration and HC sugars.

CONCLUSIONS

Differences between the CW and ND‐AD method were due to solubilized CW fractions in hot detergents.

Differences were observed in CW composition between cereals and among maturity stages that could explain differences in animal performance.

More research is warranted in order to further elucidate details of CW composition related to maturity cereal type.